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- EMDB-43667: Soluble ectodomain of human cytomegalovirus (HCMV) glycoprotein B... -

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Basic information

Entry
Database: EMDB / ID: EMD-43667
TitleSoluble ectodomain of human cytomegalovirus (HCMV) glycoprotein B (gB) in the postfusion conformation in complex with 1G2 and 7H3 Fabs
Map dataSharpened
Sample
  • Complex: Soluble ectodomain of human cytomegalovirus (HCMV) glycoprotein B (gB) in the postfusion conformation in complex with 1G2 and 7H3 Fabs
    • Protein or peptide: Envelope glycoprotein B
    • Protein or peptide: 1G2 Fab Heavy Chain
    • Protein or peptide: 1G2 Fab Light Chain
    • Protein or peptide: 7H3 Fab Light Chain
    • Protein or peptide: 7H3 Fab Heavy Chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Keywordsorthoherpesvirus / betaherpesvirus / cytomegalovirus / human betaherpesvirus 5 / human cytomegalovirus / HCMV / glycoprotein B / gB / HCMV gB / VIRAL PROTEIN / 7H3 / 1G2 / VIRAL PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


host cell Golgi membrane / host cell endosome membrane / symbiont entry into host cell / viral envelope / virion attachment to host cell / host cell plasma membrane / virion membrane / membrane
Similarity search - Function
Herpesvirus Glycoprotein B, antigenic domain, N-terminal / Glycoprotein B N-terminal antigenic domain of HCMV / Herpesvirus Glycoprotein B / Herpesvirus Glycoprotein B, PH-like domain 1 / Herpesvirus Glycoprotein B, PH-like domain 2 / Herpesvirus Glycoprotein B, PH-like domain 2 superfamily / Herpesvirus Glycoprotein B ectodomain / Herpesvirus Glycoprotein B / Herpesvirus Glycoprotein B PH-like domain
Similarity search - Domain/homology
Envelope glycoprotein B
Similarity search - Component
Biological speciesHuman betaherpesvirus 5 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsSponholtz MR / Byrne PO / McLellan JS
Funding support1 items
OrganizationGrant numberCountry
Other private
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Structure-based design of a soluble human cytomegalovirus glycoprotein B antigen stabilized in a prefusion-like conformation.
Authors: Madeline R Sponholtz / Patrick O Byrne / Alison G Lee / Ajit R Ramamohan / Jory A Goldsmith / Ryan S McCool / Ling Zhou / Nicole V Johnson / Ching-Lin Hsieh / Megan Connors / Krithika P ...Authors: Madeline R Sponholtz / Patrick O Byrne / Alison G Lee / Ajit R Ramamohan / Jory A Goldsmith / Ryan S McCool / Ling Zhou / Nicole V Johnson / Ching-Lin Hsieh / Megan Connors / Krithika P Karthigeyan / Chelsea M Crooks / Adelaide S Fuller / John D Campbell / Sallie R Permar / Jennifer A Maynard / Dong Yu / Matthew J Bottomley / Jason S McLellan /
Abstract: Human cytomegalovirus (HCMV) glycoprotein B (gB) is a class III membrane fusion protein required for viral entry. HCMV vaccine candidates containing gB have demonstrated moderate clinical efficacy, ...Human cytomegalovirus (HCMV) glycoprotein B (gB) is a class III membrane fusion protein required for viral entry. HCMV vaccine candidates containing gB have demonstrated moderate clinical efficacy, but no HCMV vaccine has been approved. Here, we used structure-based design to identify and characterize amino acid substitutions that stabilize gB in its metastable prefusion conformation. One variant containing two engineered interprotomer disulfide bonds and two cavity-filling substitutions (gB-C7), displayed increased expression and thermostability. A 2.8 Å resolution cryoelectron microscopy structure shows that gB-C7 adopts a prefusion-like conformation, revealing additional structural elements at the membrane-distal apex. Unlike previous observations for several class I viral fusion proteins, mice immunized with postfusion or prefusion-stabilized forms of soluble gB protein displayed similar neutralizing antibody titers, here specifically against an HCMV laboratory strain on fibroblasts. Collectively, these results identify initial strategies to stabilize class III viral fusion proteins and provide tools to probe gB-directed antibody responses.
History
DepositionFeb 9, 2024-
Header (metadata) releaseSep 18, 2024-
Map releaseSep 18, 2024-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43667.png

Downloads & links

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Map

FileDownload / File: emd_43667.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 512 pix.
= 426.598 Å		0.83 Å/pix.
x 512 pix.
= 426.598 Å		0.83 Å/pix.
x 512 pix.
= 426.598 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8332 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.17
Minimum - Maximum-0.64084613 - 1.0011692
Average (Standard dev.)0.00014917551 (±0.015032465)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 426.59836 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_43667_msk_1.map
Projections & Slices
AxesZYX

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Histogram

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Additional map: Unsharpened

Fileemd_43667_additional_1.map
AnnotationUnsharpened
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: Half B

Fileemd_43667_half_map_1.map
AnnotationHalf B
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: Half A

Fileemd_43667_half_map_2.map
AnnotationHalf A
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Sample components

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Entire : Soluble ectodomain of human cytomegalovirus (HCMV) glycoprotein B...

EntireName: Soluble ectodomain of human cytomegalovirus (HCMV) glycoprotein B (gB) in the postfusion conformation in complex with 1G2 and 7H3 Fabs
Components
  • Complex: Soluble ectodomain of human cytomegalovirus (HCMV) glycoprotein B (gB) in the postfusion conformation in complex with 1G2 and 7H3 Fabs
    • Protein or peptide: Envelope glycoprotein B
    • Protein or peptide: 1G2 Fab Heavy Chain
    • Protein or peptide: 1G2 Fab Light Chain
    • Protein or peptide: 7H3 Fab Light Chain
    • Protein or peptide: 7H3 Fab Heavy Chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Soluble ectodomain of human cytomegalovirus (HCMV) glycoprotein B...

SupramoleculeName: Soluble ectodomain of human cytomegalovirus (HCMV) glycoprotein B (gB) in the postfusion conformation in complex with 1G2 and 7H3 Fabs
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5

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Macromolecule #1: Envelope glycoprotein B

MacromoleculeName: Envelope glycoprotein B / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Human betaherpesvirus 5
Molecular weightTheoretical: 88.931438 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MESRIWCLVV CVNLCIVCLG AAVSSSSTRG TSATHSHHSS HTTSAAHSRS GSVSQRVTSS QTVSHGVNET IYNTTLKYGD VVGVNTTKY PYRVCSMAQG TDLIRFERNI VCTSMKPINE DLDEGIMVVY KRNIVAHTFK VRVYQKVLTF RRSYAYIHTT Y LLGSNTEY ...String:
MESRIWCLVV CVNLCIVCLG AAVSSSSTRG TSATHSHHSS HTTSAAHSRS GSVSQRVTSS QTVSHGVNET IYNTTLKYGD VVGVNTTKY PYRVCSMAQG TDLIRFERNI VCTSMKPINE DLDEGIMVVY KRNIVAHTFK VRVYQKVLTF RRSYAYIHTT Y LLGSNTEY VAPPMWEIHH INSHSQCYSS YSRVIAGTVF VAYHRDSYEN KTMQLMPDDY SNTHSTRYVT VKDQWHSRGS TW LYRETSN LNCMVTITTA RSKYPYHFFA TSTGDVVDIS PFYNGTNRNA SYFGENADKF FIFPNYTIVS DFGRPNSALE THR LVAFLE RADSVISWDI QDEKNVTCQL TFWEASERTI RSEAEDSYHF SSAKMTATFL SKKQEVNMSD SALDCVRDEA INKL QQIFN TSYNQTYEKY GNVSVFETTG GLVVFWQGIK QKSLVELERL ANRSSLNLTH NSTKSSTDGN NATHLSNMES VHNLV YAQL QFTYDTLRGY INRALAQIAE AWCVDQRRTL EVFKELSKIN PSAILSAIYN KPIAARFMGD VLGLASCVTI NQTSVK VLR DMNVKESPGR CYSRPVVIFN FANSSYVQYG QLGEDNEILL GNHRTEECQL PSLKIFIAGN SAYEYVDYLF KRMIDLS SI STVDSMIALD IDPLENTDFR VLELYSQKEL RSSNVFDLEE IMREFNSYKQ RVKYVEDKVV DPGSGYIPEA PRDGQAYV R KDGEWVLLST FLGAAASLEV LFQGPGHHHH HHHHSAWSHP QFEKGGASGG GGSGGSAWSH PQFEK

UniProtKB: Envelope glycoprotein B

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Macromolecule #2: 1G2 Fab Heavy Chain

MacromoleculeName: 1G2 Fab Heavy Chain / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.206172 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QLQLQESGPG LVKPSETLSL TCTVSGASID RSTYYWGWIR QPPGKGLEWI ANIYYNGRAV YSPSLKSRVT ISVDTSKNQF SLKVRSLTA ADTAVYYCAT RWNYFFDFDY WGRGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV S WNSGALTS ...String:
QLQLQESGPG LVKPSETLSL TCTVSGASID RSTYYWGWIR QPPGKGLEWI ANIYYNGRAV YSPSLKSRVT ISVDTSKNQF SLKVRSLTA ADTAVYYCAT RWNYFFDFDY WGRGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV S WNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKKV EPKSCD

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Macromolecule #3: 1G2 Fab Light Chain

MacromoleculeName: 1G2 Fab Light Chain / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 22.853125 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QSVLTQPPSA SGTPGQRVTI SCSGSSSNIE TNYVSWYQQF PGTAPKLLIY RNNQRPSGVP DRFSGSKSGT SASLAISGLR SEDEAEYYC GTWDDNSWVF GGGTKLTVLG QPKAAPSVTL FPPSSEELQA NKATLVCLIS DFYPGAVTVA WKADSSPVKA G VETTTPSK ...String:
QSVLTQPPSA SGTPGQRVTI SCSGSSSNIE TNYVSWYQQF PGTAPKLLIY RNNQRPSGVP DRFSGSKSGT SASLAISGLR SEDEAEYYC GTWDDNSWVF GGGTKLTVLG QPKAAPSVTL FPPSSEELQA NKATLVCLIS DFYPGAVTVA WKADSSPVKA G VETTTPSK QSNNKYAASS YLSLTPEQWK SHRSYSCQVT HEGSTVEKTV APTECS

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Macromolecule #4: 7H3 Fab Light Chain

MacromoleculeName: 7H3 Fab Light Chain / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 22.879361 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QSVLSQPPSA SGTPGQRVTI SCSGSSSNIG KNYVYWYQQV PGTAPKLLMF KNNQRPSGVP DRFSGSKSGT SASLAISGLR SEDEADYYC SAWDGSLSRP LFGGGTKVTV LGQPKAAPSV TLFPPSSEEL QANKATLVCL ISDFYPGAVT VAWKADSSPV K AGVETTTP ...String:
QSVLSQPPSA SGTPGQRVTI SCSGSSSNIG KNYVYWYQQV PGTAPKLLMF KNNQRPSGVP DRFSGSKSGT SASLAISGLR SEDEADYYC SAWDGSLSRP LFGGGTKVTV LGQPKAAPSV TLFPPSSEEL QANKATLVCL ISDFYPGAVT VAWKADSSPV K AGVETTTP SKQSNNKYAA SSYLSLTPEQ WKSHRSYSCQ VTHEGSTVEK TVAPTECS

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Macromolecule #5: 7H3 Fab Heavy Chain

MacromoleculeName: 7H3 Fab Heavy Chain / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.152064 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QVQLVQSGAE VKNPGASVKV SCKASGYTFT DYYIHWVRQA PGQGLEWMGW FNPNSGGTNF VQNFQGRVTM TRDTSISTAY MELSRLRSD DTAMYYCAKD SAKTASAYYG LNFFYYGMDV WGQGTTVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV K DYFPEPVT ...String:
QVQLVQSGAE VKNPGASVKV SCKASGYTFT DYYIHWVRQA PGQGLEWMGW FNPNSGGTNF VQNFQGRVTM TRDTSISTAY MELSRLRSD DTAMYYCAKD SAKTASAYYG LNFFYYGMDV WGQGTTVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV K DYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKKV EPKSCD

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Macromolecule #8: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 8 / Number of copies: 10 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3 mg/mL
BufferpH: 8
Details: 2 mM Tris pH 8.0, 200 mM NaCl, 0.02% (w/v) sodium azide, 3% (v/v) glycerol, 0.25% CHAPS, and 0.1% (w/v) amphipol A8-35
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsSoluble ectodomain of human cytomegalovirus (HCMV) glycoprotein B (gB) in the postfusion conformation, 1G2 Fab, and 7H3 Fab purified separately, mixed, and incubated for 30 minutes on ice prior to grid preparation

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 8679 / Average electron dose: 80.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.0) / Number images used: 211132
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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